Project Summary/Abstract Acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN) are the most common myeloid malignancies in adult patient population. Stem cell transplantation remains the most curative option as only a fraction of these patients is cured with standard chemotherapy regimens. Recent advances using post-transplant cyclophosphamide (PTCy) pioneered by researchers at John?s Hopkins University have significantly improved outcomes after HLA haploidentical donor transplantation (haplo-HCT), expanding donor availability for patients who otherwise lack traditionally matched donors. However, relapse is the most common cause of treatment failure after stem cell transplantation including in haplo-HCT patients. Furthermore, post-transplant relapsed patients have extremely poor prognosis with the current treatment approaches including donor lymphocyte infusion (DLI); novel therapies are a major unmet need. We and others recently described cytokine induced memory-like (CIML) NK cells with superior activity against leukemia cells. CIML NK cells were safe and demonstrated very promising activity (>50% compete remission rates) in our recently completed early phase trial in patients with advanced AML and MDS in a non-transplant setting. In the current study we will attempt to harness enhanced anti-leukemia activity of CIML NK cells to treat patients who have relapsed after haplo-HCT and where donor-derived immunologic environment permits extended activity and survival of infused cells. In Specific Aim 1, we will assess safety and possible efficacy of CIML NK cells in patients with myeloid malignancies relapsed after haplo-HCT. Peripheral blood mononuclear cells will be harvested from the original stem cell donors using non-mobilized leukapheresis and NK cells separated using two step CD3 depletion followed by CD56 positive selection. CIML NK cells will be generated from the conventional NK cells by overnight activation with cytokines (IL-12/IL-15/IL-18) and infused into the patients after they receive lymphodepleting Flu/Cy chemotherapy. Afterwards, the patients will receive low dose IL-2 (1 x106 IU/m2 SQ every other day x7 doses) to further help support / activate adoptively transferred CIML NK cells in vivo. In Specific Aim 2, we will sample peripheral blood (PB) and bone marrow (BM) to characterize immune subsets in detail, track the persistence and functional status of transferred CIML cells and evaluate the tumor immune microenvironment at the single cell resolution. The proposal incorporates strong preliminary data, compelling biologic rationale, clinical feasibility, and major therapeutic impact. The results from this study will potentially help develop a new safer viable treatment option for patients with post-transplant relapsed myeloid malignancies and help advance our knowledge regarding key aspects of the in vivo CIML NK cell biology.